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• W2129582005 endingPage "1941" @default.
• W2129582005 startingPage "1933" @default.
• W2129582005 abstract "Elucidation of post-translational modifications to proteins, such as glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity. Elucidation of post-translational modifications to proteins, such as glycosylations or phosphorylations, is one of the major issues concerning ongoing proteomics studies. To reduce general sample complexity, a necessary prerequisite is specific enrichment of peptide subsets prior to mass spectrometric sequencing. Regarding analysis of overall N-glycosylation sites in the past, this has been achieved by several approaches proving to be more or less complicated and specific. Here we present a novel strategy to target N-glycosylation sites with application to platelet membrane proteins. Initial aqueous two-phase partitioning for membrane enrichment and single step strong cation exchange-based purification of glycopeptides resulted in identification of 148 glycosylation sites on 79 different protein species. Although 69% of these sites were not annotated in the Swiss-Prot database before, a high number of 75% plasma membrane-localized proteins were analyzed. Furthermore miniaturizations and relative quantification are comprised in the developed method suggesting further use in other proteome projects. Results on platelet glycosylation sites may imply an impact on research of bleeding disorders as well as potential new functions in inflammation and immunoactivity. During recent years, the fields of proteomics and glycomics have experienced rapid advancements both in analytical study design as well as instrumental setup. Besides common proteomics experiments monitoring complete cells or tissues, e.g. by differential two-dimensional PAGE, current focus has turned to subproteomes or specific subsets of proteins. Glycomics has emerged as a field of growing interest due to widespread importance of carbohydrate additions to proteins. Glycosylations are divided into several classes, such as N-glycosylation, O-glycosylation (1Hanisch F.G. O-Glycosylation of the mucin type.Biol. Chem. 2001; 382: 143-149Crossref PubMed Scopus (272) Google Scholar), glycosylphosphatidylinositol anchors, C-mannosylation (2Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), etc., as well as a high number of structural isoforms. Therefore, many strategies have focused on distinct subsets of glycoproteins or -peptides. To analyze glycan attachment sites, several methods have been reported in the past. Carbohydrate-lectin interactions have been extensively used for isolation of glycopeptides and subsequent site analysis (3Kaji H. Saito H. Yamauchi Y. Shinkawa T. Taoka M. Hirabayashi J. Kasai K. Takahashi N. Isobe T. Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins.Nat. Biotechnol. 2003; 21: 667-672Crossref PubMed Scopus (570) Google Scholar). Moreover trapping by hydrazide chemistry (4Zhang H. Li X.J. Martin D.B. Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.Nat. Biotechnol. 2003; 21: 660-666Crossref PubMed Scopus (1275) Google Scholar) as well as enrichment of glycopeptides by normal phase chromatography has been demonstrated (5Hagglund P. Bunkenborg J. Elortza F. Jensen O.N. Roepstorff P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation.J. Proteome Res. 2004; 3: 556-566Crossref PubMed Scopus (410) Google Scholar). As shown with platelets, hydrazide chemistry and lectin affinity approaches are readily applicable to these anucleate cells (6Lewandrowski U. Moebius J. Walter U. Sickmann A. Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.Mol. Cell. Proteomics. 2006; 5: 226-233Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In this context, former proteome studies on platelets revealed that only a small proportion of membrane-bound proteins are accessible by traditional proteomics techniques such as two-dimensional PAGE coupled with mass spectrometry (7Garcia A. Prabhakar S. Brock C.J. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Zitzmann N. Extensive analysis of the human platelet proteome by two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2004; 4: 656-668Crossref PubMed Scopus (149) Google Scholar, 8O’Neill E.E. Brock C.J. von Kriegsheim A.F. Pearce A.C. Dwek R.A. Watson S.P. Hebestreit H.F. Towards complete analysis of the platelet proteome.Proteomics. 2002; 2: 288-305Crossref PubMed Scopus (172) Google Scholar, 9Marcus K. Immler D. Sternberger J. Meyer H.E. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins.Electrophoresis. 2000; 21: 2622-2636Crossref PubMed Scopus (174) Google Scholar). Therefore, subfractionation is a prerequisite to cover the high dynamic range of protein isoforms within platelets. For the identification of N-glycosylation sites especially on platelet membrane proteins we focused on aqueous two-phase partitioning for membrane enrichment and specific purification of a glycopeptide subset by strong cation exchange (SCX) 1The abbreviations used are: SCX, strong cation exchange; ENSAS, enhanced N-glycosylation site analysis using strong cation exchange enrichment; PEG, polyethylene glycol; PNGaseF, peptide:N-glycosidase F. chromatography. Recently the method of polymer-based two-phase partitioning gained interest in the field of proteomics due to its simplicity and reliability (10Everberg H. Peterson R. Rak S. Tjerneld F. Emanuelsson C. Aqueous two-phase partitioning for proteomic monitoring of cell surface biomarkers in human peripheral blood mononuclear cells.J. Proteome Res. 2006; 5: 1168-1175Crossref PubMed Scopus (19) Google Scholar, 11Schindler J. Lewandrowski U. Sickmann A. Friauf E. Nothwang H.G. Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning.Mol. Cell. Proteomics. 2006; 5: 390-400Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 12Cao R. Li X. Liu Z. Peng X. Hu W. Wang X. Chen P. Xie J. Liang S. Integration of a two-phase partition method into proteomics research on rat liver plasma membrane proteins.J. Proteome Res. 2006; 5: 634-642Crossref PubMed Scopus (73) Google Scholar). Most commonly polyethylene glycol/dextran mixtures are used to generate a system comprising 1) a polyethylene glycol-rich upper phase and 2) a lower phase enriched with dextran (13Persson A. Jergil B. The purification of membranes by affinity partitioning.FASEB J. 1995; 9: 1304-1310Crossref PubMed Scopus (25) Google Scholar). Upon careful choice of parameters such as salt and polymer concentration, plasma membranes tend to partition preferentially to the upper phase due to their physicochemical properties (14Albertsson P.-A. Partition of cell particles and macromolecules.in: Albertsson P.-A. Partition of Cell Particles and Macromolecules. Wiley Interscience, New York1971: 233-242Google Scholar). Thereby they are separated from the bulk of intracellular membranes, e.g. endoplasmic reticulum and mitochondria. However, in the case of platelets a strict partitioning is limited due to their unique cellular morphology comprising e.g. a dense tubular membrane system. Nevertheless the method has the advantage of rapid and gentle sample preparation. It can also be easily scaled in respect to available sample amounts. Although membrane preparations are already enriched in glycoproteins, they still contain high numbers of non-glycosylated proteins, e.g. high abundant structural components like actin. A further purification of plasma membrane glycopeptides is necessary to assess N-glycosylation sites in larger scale by avoiding suppression effects during mass spectrometric analysis. In contrast to already mentioned glycopeptide enrichment methods based on specific trapping of glycopeptide subsets, SCX enrichment increases the relative amount of glycopeptides by trapping and removing all non-glycopeptides in the sample. The principle of this novel approach (see Fig. 1) is based on the assumption that the majority of extracellular N-glycans contains sialic acid residues, which reduce the positive net charge of glycopeptides in comparison with other peptides. At low pH the majority of tryptic peptides are theoretically doubly and triply charged because positive charges can be attributed to the N terminus and to C-terminal lysine or arginine residues, respectively. Sialylated glycopeptides containing additional negative charges are therefore eluted prior to non-glycopeptides using strong cation exchange material. A similar principle has also been applied for the isolation of phosphopeptides by strong cation exchange chromatography (15Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1237) Google Scholar) in the past. In this work we devised a novel strategy by combining for the first time principles of aqueous two-phase partitioning and strong cation exchange chromatography for enhanced N-glycosylation site analysis using strong cation exchange enrichment (ENSAS). Applied to human platelets ENSAS led to the identification of 148 individual glycosylation sites on 79 proteins. Thereof 102 sites (69%) were not previously described in the Swiss-Prot database, and a high proportion of 75% plasma membrane-associated proteins was observed, e.g. low abundance G-protein-coupled receptors. Furthermore several new platelet proteins were found in this study with implications on current platelet research and potential clinical relevance. All chromatographic chemicals used in this study were obtained from Sigma as analytical or higher grade. PNGaseF (Flavobacterium meningosepticum) and neuraminidase (Clostridium perfringens) were from Roche Applied Science. Sequencing grade trypsin was purchased from Promega, Madison, WI. Human platelets were prepared by differential centrifugation and washing steps with minor modifications as described previously (6Lewandrowski U. Moebius J. Walter U. Sickmann A. Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.Mol. Cell. Proteomics. 2006; 5: 226-233Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 16Tandon N.N. Lipsky R.H. Burgess W.H. Jamieson G.A. Isolation and characterization of platelet glycoprotein IV (CD36).J. Biol. Chem. 1989; 264: 7570-7575Abstract Full Text PDF PubMed Google Scholar). Briefly fresh apheresis-derived and already leukocyte-depleted platelets (Department of Transfusion Medicine, University Würzburg, Germany) were centrifuged twice at 310 × g for 15 min to remove remaining leukocytes or erythrocytes. The platelet-containing supernatant was centrifuged at 380 × g for 20 min at room temperature, and pelleted platelets were washed twice with 10 mm citric acid buffer containing 5 mm KCl, 145 mm NaCl, 14 mm glucose, and 1 mm MgCl2, pH 6.4, to remove residual plasma proteins from the apheresis preparation. The obtained platelet pellets were frozen in liquid nitrogen until further use. For the enrichment of platelet plasma membranes two-phase partitioning as a variation of previously published methods was used (11Schindler J. Lewandrowski U. Sickmann A. Friauf E. Nothwang H.G. Proteomic analysis of brain plasma membranes isolated by affinity two-phase partitioning.Mol. Cell. Proteomics. 2006; 5: 390-400Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). A 200-ml two-phase system consisting of 6.3% PEG 3350 and dextran T500 each in 15 mm Tris, pH 7.8, was prepared and equilibrated overnight at 6 °C. Furthermore a 20-ml system (1:1 (v/v) of the equilibrated PEG and dextran phases) was used for separation of 100 mg (wet weight) of platelets. Lysis was performed by repetitive ultrasonic bursts, and subsequent phase separation was achieved by centrifugation at 500 × g for 10 min at 6 °C. The top PEG phase was removed and mixed with a fresh dextran phase followed by gentle mixing. This exchange was repeated once, and the final upper PEG phase was diluted 1:1 with water before membranes were pelleted by ultracentrifugation at 100,000 × g for 1 h at 4 °C in a TLA 100.4 rotor (Beckman Coulter, Krefeld, Germany). The resulting four pellets were subjected to 2-fold carbonate extractions in a volume of 3 ml of 100 mm sodium carbonate, pH 11.5, each as described elsewhere (45Fujiki Y. Kubbard A.L. Fowler S. Lazarow P.B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum.J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1383) Google Scholar). A single pellet (or an equivalent amount of intact platelets) was solubilized in 200 μl of 2.5% SDS, 25 mm NH4HCO3, pH 7.8, and 1,4-dithiothreitol was added to a concentration of 5 mm prior to incubation at 57 °C for 15 min. Upon addition of iodoacetamide to a final concentration of 20 mm and incubation at 21 °C for an additional 15 min, 1800 μl of ethanol was added, and proteins were precipitated at −30 °C for 4 h for removal of SDS. Proteolytic digestion of solubilized proteins in 400 μl of NH4HCO3 with trypsin (sequencing grade, Promega) was performed at 37 °C overnight. For removal of undigested proteins, samples were ultrafiltrated with a-10 kDa molecular mass cutoff (Amicon Ultrafree-MC, Millipore, Schwalbach, Germany). For HPLC enrichment of glycopeptide-containing fractions, a 2.1-mm-inner diameter × 15-cm long column (PolySULFOETHYL Aspartamide, 200-Å pore size, 5-μm particle size; Chromatographic Technologies, Basel, Switzerland) in combination with a Famos™/Ultimate™ HPLC system (Dionex, Idstein, Germany) was used. A binary buffer system consisting of 5 mm NaH2PO4, pH 2.7 (buffer A) and 5 mm NaH2PO4, 15% acetonitrile, 500 mm NaCl, pH 2.7 (buffer B) was used to equilibrate the column at 1% B and a flow rate of 120 μl prior to separation. Upon injection of 100-μl sample aliquots, the column was washed for 7 min at 1% B followed by a gradual increase to 30% B (28 min) and 99% B (29 min). After rinsing the column for 7 min, the system was equilibrated to 1% B again. Fractions were collected in 60-s intervals by a ProteineerFC fraction collector (Bruker Daltonics, Bremen, Germany). Self-made spin columns were constructed using C18 pipette tips (Omix minibed; Varian, Darmstadt, Germany) packed with a 5-μl volume of SCX material (see “SCX Prefractionation”) that were placed within HPLC borosilicate sampling vials (CS-Chromatographie, Langerwehe, Germany). Spin columns were washed three times with 30 μl of 5 mm NaH2PO4, 50% acetonitrile, pH 2.7, at 500 × g. Samples were freeze-dried, solubilized in 30 μl of washing buffer, and added on top of the columns; upon slow centrifugation at 500 × g the flow-through was collected, and columns were subsequently discarded to avoid cross-contamination with further samples. Prior to SCX enrichment, 20-μl peptide digest aliquots were diluted with 120 μl of 50 mm ammonium acetate, pH 5.0, and treated with 0.08 unit of neuraminidase for 16 h at 37 °C. To remove glycan side chains after SCX enrichment, digests with PNGaseF were performed at 5 units/100-μl fraction size in 50 mm ammonium hydrogen carbonate, pH 7.8, for 16 h at 37 °C. Separation of peptide mixtures prior to mass spectrometric sequencing was achieved by nano-LC-MS/MS coupling. A Famos, Switchos™, Ultimate nano-LC system (Dionex) was used to trap and desalt isolated peptide mixtures on a self-made 100-μm-inner diameter × 2-cm long precolumn (Ace C18, 5-μm particle size, 10-Å pore size; HiChrom Ltd., Berkshire, UK) with 0.1% TFA as loading buffer. Separation on a self-made 75-μm-inner diameter × 150-mm long separation column (Ace C18, 3-μm particle size, 100-Å pore size; HiChrom Ltd.) was performed at a flow rate of 270 nl/min and gradient slopes of 1% B/min and 0.5%B/min up to 55% B content, respectively. Solvent A was 0.1% formic acid in water, and solvent B was 0.1% formic acid in 84% acetonitrile. A Qtrap 4000 linear ion trap mass spectrometer was used in the positive ion mode comprising 1) an enhanced multiple charge scan (380–1500 amu, three spectra summed at 4000 amu/s) as survey scan followed by 2) enhanced resolution scans of selected precursors (single spectra at 250 amu/s) that were furthermore sequenced by 3) enhanced product ion scans (115–1500 amu, two spectra summed at 4000 amu/s). Ion spray voltage was set to 2.3 kV, and only ions with charge states 2+ and 3+ were chosen for fragmentation. Dynamic exclusion time was set to 22 s after one occurrence of a respective target ion. To further separate peptide mixtures, two independent procedures were added to the described analysis pathway: 1) on-line two-dimensional fractionation by SCX/reversed phase coupling and 2) gas phase fractionation in combination with 0.5% B/min reversed phase gradients. For SCX on-line fractionation a triphasic precolumn (100-μm inner diameter, 1.5-cm C18/1.5-cm SCX/1.5-cm C18) was used. Samples were applied in 0.1% formic acid and flushed onto the first C18 phase. Upon elution to the SCX phase by a 4% B/min gradient, peptide subsets were stepwise eluted onto the second C18 phase by injection of 1 μl of 1, 5, 7.5, 10, 20, 30, 50, 70, 90, and 150 mm ammonium acetate, pH 2.7. Common reversed phase gradients as described above were used to separate individual subsets of peptides. Because the sample amount was not severely limited gas phase fractionation was applied by limiting the range of survey scans to smaller individual intervals (380–500 m/z, 490–600 m/z, 590–700 m/z, 690–900 m/z, and 890–1500 m/z, all measured at 4000 amu/s with five spectra summed). Thereby the same sample was measured five times with triple play events as described above, fragmenting the three most intensive ions in the respective m/z intervals at a time. Mass spectrometry-derived datasets were evaluated by database searches using the Mascot™ search algorithm (Version 2.1, Matrix Science, London, UK). Peak lists were generated from the raw data format using Analyst 1.4 software plug-ins (mascot.dll; Matrix Science/Applied Biosystems). All peaks with intensities below 0.1% of the base peak were omitted while data were centroided in the process. Mass deviance was set to 0.4 Da to readily identify asparagine to aspartic acid conversion of +1 Da. A non-redundant human subset of the Swiss-Prot database (version from May 31, 2006; total 222,289 sequences, thereof 14,106 in the human subset; www.expasy.ch) was used for searches with trypsin specified as protease comprising one missed cleavage site. Spectra with a Mascot score >35 (significance threshold p < 0.05) and valid glycosylation consensus sequence were considered for further manual evaluation. The Multi-Protein Survey System (Shanghai Center for Bioinformation Technology) was used for data evaluation and assignment of protein function (see Supplemental Table 1). For prediction of transmembrane domains TMHMM 2.0 was used (Center for Biological Sequence Analysis, Technical University of Denmark). Purification of platelet plasma membranes by aqueous two-phase partitioning proved to be an efficient method for sample preparation prior to ENSAS enrichment. The complete procedure is readily performed in 4–5 h. Preliminary results of one-dimensional SDS-PAGE coupled with nano-LC-MS/MS sequencing indicated over 200 different proteins with a high percentage of integral membrane proteins (data not shown). This finding also applies to proteins identified in the current study. Of 79 identified proteins (Table I), 89% could be assigned to membranes of organelles such as plasma membrane (64%), or membrane localization was predicted based upon primary sequence analysis but with unknown localization so far (14%) (Fig. 2). In addition, proteins that are present within both the plasma membrane and other organelles (Golgi, endosomes, etc.) could be identified (9%).Table IComplete list of analyzed glycoproteinsAccession no.NameNo.O14672ADA101O15031PLXB23O60449LY753O75954TSN91O95497VNN12O95857TSN131O95858TSN151O95866G6B1P01009A1AT1P01137TGFB11P02675FIBB1P02751FINC1P04156PRIO1P04275VWF2P05106ITB33P05556ITB12P06756ITAV2P07359GP1BA1P08174DAF1P081954F23P08575CD451P08648ITA56P08962CD633P10909CLUS2P11279LAMP15P13598ICAM24P13987CD591P15529MCP1P16109LYAM32P16284PECA15P16671CD364P17301ITA23P19256LFA31P19440GGT11P20645MPRD1P23229ITA63P25942TNR51P27701CD821P28906CD341P30825CTR11P34810CD681P35613BASI2P40189IL6RB3P40197GPV2P42892ECE14P43119PI2R1P49641MA2A21P51575P2RX12P51679CCR41P53801PTTG1P54709AT1B31P54852EMP31P78536ADA171Q04771ACVR11Q07108CD691Q08722CD472Q12913PTPRJ8Q13201MMRN18Q13308PTK71Q14108SCRB21Q14118DAG12Q15762CD2262Q16563SYPL11Q4KMQ2TM16F2Q8IVB4SL9A91Q8IWA5CTL22Q8TCT8PSL22Q8WWI5CTL11Q96F46I17RA1Q96RD7PANX11Q96RI0PAR41Q99523SORT1Q9HD45TM9S31Q9NXL6SIDT11Q9NY35CLDND1Q9UIQ6LCAP1Q9Y210TRPC62Q9Y639NPTN1Q9Y6C2EMIL12 Open table in a new tab Because the complete strategy uses no gel-based separation, suitability for purification of membrane proteins is achieved; initially proteins are still embedded in biological membranes, and later on the detergent is removed directly before the proteolytic digest, which in turn produces smaller and thus more soluble peptides. The GRAVY index, an indicator for the hydrophobicity of identified protein sequences (see Supplemental Table 1), shows a broad scattering of values ranging from hydrophilic and abundant proteins such as fibrinogen (−0.78) over low abundance membrane receptors such as proteinase-activated receptor 4 (+0.41) to extremely hydrophobic proteins such as the tetraspanin family with tetraspanin 13 (+0.81). In general, numerous proteins with multiple membrane-spanning domains were detected; examples are the G-protein-coupled receptor PAR4 with seven predicted transmembrane domains and TM9S3 with nine or SID1 with 11 predicted membrane-spanning domains. In the current approach plasma membrane glycoproteins as potential initiators of vital platelet functions were targeted. Therefore, strong cation exchange chromatography was used for enrichment of sialylated glycopeptides. These peptides were collected in flow-through fractions with low contaminations by non-glycopeptides. Of 595 peptides identified by Mascot with a score above 35, 83% contained a modified asparagine within the N-glycosylation consensus sequence. Remaining peptides either had no modification at all or were modified at asparagines outside of the consensus sequences. However, the majority of these spectra were of inferior quality regarding noise levels and signal assignment by the algorithm. Several glycopeptides kept eluting after 10 min, although this observation was attributed to smearing effects because those peptides were identified in several adjacent fractions. At later gradient steps, no distinct signals of glycopeptides could be identified probably due to quenching effects by the surpassing number of non-glycopeptides. To clarify the mechanism of sialoglycopeptide enrichment during SCX chromatography, a neuraminidase with broad specificity cleaving α(2–3)-, α(2–6)-, and α(2–8)-bound N-acetylneuraminic acid was used to remove potential negative charges from glycopeptides. This cleavage allows for answering whether the non-retention of glycopeptides is due to additional negative charges or to potential size exclusion effects. It was shown by Alvarez-Manilla et al. (17Alvarez-Manilla G. Atwood III, J. Guo Y. Warren N.L. Orlando R. Pierce M. Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites.J. Proteome Res. 2006; 5: 701-708Crossref PubMed Scopus (171) Google Scholar) that glycopeptides may also be enriched by size exclusion chromatography. Furthermore the SCX resin used in the current work has been demonstrated as a size exclusion stationary phase elsewhere (18Alpert A.J. Size exclusion high-performance liquid chromatography of small solutes.in: Wu C.S. Column Handbook for Size Exclusion Chromatography. Academic Press, San Diego, CA1999: 249-266Crossref Google Scholar). After the neuraminidase treatment, desialylated samples exhibited no significant enrichment of glycopeptides. Only six glycosylations sites could be assigned within the samples (Asn-478/P05106, Asn-37/P07359, Asn-198/P27701, Asn-344,Asn-356/P16284, Asn-374/P10909) including two sites for the high abundance proteins integrin β3 and platelet glycoprotein Ib α chain. It was concluded that these identifications can be attributed to partially incomplete desialylation. Therefore, desialylated peptides seem to be as strongly retarded as their unglycosylated counterparts that elute later in the gradient. Furthermore size exclusion effects on ENSAS seem improbable due to the results of the neuraminidase treatment. Although the loss of sialic acids would reduce the size of glycopeptides, such a complete reduction of the enrichment effect seems improbable in respect to the remaining carbohydrate moieties. By targeting sialoglycopeptides of membrane glycoproteins, a total of 148 individual glycosylation sites distributed to 79 proteins could be identified (Table I). Upon comparing the sites with the Swiss-Prot database, 69% have not been determined previously (Fig. 2). Thereof 94 sites or 63% were only automatically annotated as potential glycosylation sites based on the presence of NX(S/T) consensus sequences. However, no experimental proof was provided in those cases so far. In comparison with former hydrazide and lectin affinity procedures of whole platelet lysates only a minor overlap of 16 sites (11%) was noted. Those sites contribute to the 46 hits (31%) termed as ‘known’ in the current study. Because the focus of ENSAS enrichment is set selectively to glycopeptides, it results in a dramatic reduction of sample complexity. Thereby the unfavorable balance of predominant identification of high abundance proteins in proteomics is shifted in favor of those with very low abundance. Of the 79 identified proteins, 46 were identified based on single glycopeptides, and a further 18 proteins were identified by two glycopeptides. Among the former were three G-protein-coupled receptors (PAR4, CCR4, and PI2R; see also Fig. 3 for spectrum of CCR4) with a few hundred copies per cell as well as a novel glycosylation site on a lesser known platelet receptor, G6b (19Gevaert K. Goethals M. Martens L. Van Damme J. Staes A. Thomas G.R. Vandekerckhove J. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.Nat. Biotechnol. 2003; 21: 566-569Crossref PubMed Scopus (505) Google Scholar). Those sites could be found despite the high background of e.g. ITB3 with up to 80,000 copies per cell (20Clemetson K. Platelet receptors.in: Michelson A. Platelets. Academic Press, San Diego, CA2002: 65-84Google Scholar). The reduction of complexity due to the focus on sialoglycopeptides will most often result in a loss of isoform resolution due to the reduced sequence coverage of the proteins. However, tumor necrosis factor receptor subfamily member 5 was identified by a single glycosylation site directly within an isoform-determining sequence. Although the membrane-bound isoform I contains an NKT consensus sequence within the peptide KìDLVVQQAGTNKìT, there is no such consensus motif at all in the soluble isoform II. Glycosylation site analysis may therefore help in individual cases to resolve such specific isoforms that may possibly not be detected in standard proteomics experiments. The identification of N-glycosylation sites by mass spectrometry most often relies on the specific deamidation of asparagine to aspartic acid within the consensus sequence NX(S/T) (where X is not proline) upon cleavage of the glycan moiety by PNGaseF (21Tarentino A.L. Gomez C.M. Plummer Jr., T.H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.Biochemistry. 1985; 24: 4665-4671Crossref PubMed Scopus (917) Google Scholar). In general, an artificial 1-Da mass shift from asparagine to aspartic acid as enzymatically introduced by the ENSAS procedure is not u" @default.
• W2129582005 created "2016-06-24" @default.
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• W2129582005 date "2007-11-01" @default.
• W2129582005 modified "2023-10-11" @default.
• W2129582005 title "Enhanced N-Glycosylation Site Analysis of Sialoglycopeptides by Strong Cation Exchange Prefractionation Applied to Platelet Plasma Membranes" @default.
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